Tanaji K. Dongre

Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation

A battery of enzymes from the eukaryotic antioxidant defense system was measured in salivary gland and in whole body extract of fourth instar larvae of Chironomus ramosus with an objective of finding any clue for the dipteran insects capacity to tolerate heavy doses of ionizing radiation. Levels of activity of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSH-Px) were quantified in 30 days old larvae exposed to LD(20) dose of gamma radiation. Compared to controls, activity of Cu,Zn-SOD increased 3 to 4 fold and catalase 2 fold in response to ionizing radiation stress, while activities of GR and GSH-Px enzymes were decreased. Among the other SOD isoenzymes, our results showed comparable levels of Mn-SOD and Cu,Zn-SOD activity in control and irradiated groups of larvae. The increase in levels of the Cu,Zn-SOD isoenzyme was also confirmed by Western blot and zymography supported by densitometric quantification. No evidence of Fe-SOD was found in C. ramosus larvae. These findings could help to explain the persistence of natural populations of Chironomus in radioactively contaminated regions.

Purification and characterization of mosquitocidal Bacillus sphaericus BinA protein

Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal binary toxin during sporulation. The binary toxin is composed of toxic BinA (41.9kDa) and receptor binding BinB (51.4kDa) polypeptides and is active against vectors of filariasis, encephalitis and malaria. The toxin has been tested with limited use for the control of vector mosquitoes for more than two decades. The binA gene from a local ISPC-8 strain of B. sphaericus that is highly toxic to Culex and Anopheles mosquito species was cloned into pET16b and expressed in Escherichia coli. The purified BinA protein differs by one amino acid (R197M) from BinA of the highest toxicity strains 1593/2362/C3-41. Majority of the expressed protein was observed in inclusion bodies. BinA inclusions alone from E. coli did not show toxic activity, like reported previously. However, the active form of BinA could be purified to homogeneity from the soluble fraction of E. coli cell lysate, grown at reduced temperature after isopropyl beta-d-thiogalactopyranoside induction. The purified BinA protein with and without poly-histidine tag showed LC(50) dose of 82.3 and 66.9ngml(-1), respectively, at 48h against Culex quinquefasciatus larvae. The secondary structure of BinA is expected to be mainly beta strands as estimated using far-UV circular dichroism. The estimates matched well with the secondary structure predictions using amino acid sequence. This is the first report of large-scale purification and accurate toxicity estimation of soluble B. sphaericus BinA. This can help in design and synthesis of improved bacterial insecticide.

Effect of ultraviolet radiation on spore viability and mosquitocidal activity of an indigenous ISPC-8 Bacillus sphaericus Neide strain.

Effects of UV-A, UV-B and their combination on spore viability and larvicidal activity of an indigenous isolate of Bacillus sphaericus Neide, ISPC-8 were studied under laboratory conditions. The UV sensitivity of ISPC-8 was compared with standard strain 1593 and larvicidal activity was tested against third instar larvae of mosquito, Culex quinquefasciatus Say. No significant adverse effects on viability as well as larvicidal activity of both strains were observed when spores were exposed to UV-A for 6h. However, exposure to UV-B for a few minutes adversely affected the spore viability as well as larvicidal activity and this adverse effect was more pronounced on spore viability. In both strains about 50% larvicidal activity was retained after exposure of the spores to UV-B for 8h. However, spore viability at this exposure of time was drastically reduced to 2.5% in ISPC-8 and 0.3% in 1593. The spore viability and larvicidal activity patterns were found to be similar to UV-B treatment when spores were exposed to a combination of UV-A and UV-B. Our study hence, shows the adverse effect of UV radiation on ISPC-8 and 1593 indicating the need to incorporate eco-friendly UV protectants in formulations so that the efficacy of biopesticides based on these entomopathogens can be prolonged under field conditions, especially in tropical countries.

UV protectants for the biopesticide based on Bacillus sphaericus Neide and their role in protecting the binary toxins from UV radiation

The UV protectant properties of 26 natural and synthetic compounds were investigated for a biopesticide based on an indigenously isolated strain (ISPC-8) of Bacillus sphaericus Neide. In initial screening, spores of ISPC-8 with 0.1% (w/w for solid and v/w for liquid materials) concentration of different compounds were exposed to UV-B radiation (4.9 x 10(5) J/m(2)) for 6h and their spore viability and larvicidal activity were studied. The larvicidal activity was evaluated against third-instar larvae of Culex quinquefasciatus Say. There was a complete loss of spore viability (1.4% viable spores) and partial reduction in larvicidal activity (57.7% of original activity) after exposure of spores to UV-B for 6h. However, spore viability as well as larvicidal activity protected significantly when spores were mixed with different compounds before exposing them to UV-B. Among the different compounds tested benzaldehyde, congo red, para-aminobenzoic acid (PABA) and cinnamaldehyde were found to be promising in protecting the spores from UV-B radiation. The presence of binary toxins (41.9 kDa and 51.4 kDa) in protected and unprotected samples were examined by SDS-PAGE. The binary toxin bands disappeared in unprotected spores after 24h of exposure to UV-B, whereas toxin bands were distinctly visible when spores with benzaldehyde and cinnamaldehyde were exposed to UV-B for 96 h and 120 h, respectively. Congo red and PABA were found to be most effective in protecting binary toxins even after 168 h of exposure to UV-B. Incorporation of these promising UV protectant compounds in biopesticides would help in protecting the spores from the adverse effects of UV radiation and prolong the persistence of biopesticides under field conditions.

Hsp70 expression in Chironomus ramosus exposed to gamma radiation

Purpose: A tropical species of midge, Chironomus ramosus has been recently reported to be one of the radio-tolerant groups of organisms. The present study was undertaken to examine the protein profile and expression of Heat shock protein-70 (Hsp70) in gamma radiation stress, which has also been reported as a common biomarker for different type of stressors. Materials and methods: Metabolic labelling of salivary gland (SG) proteins with [35S]-methionine showed over-expression of a 70 kDa protein band up to 4 hours (h) of observation in the post exposure recovery period. For confirmation of the expression of Hsp70 in SG cells after gamma radiation exposure, semi-quantitative real-time-polymerase chain reaction (RT-PCR), Western blotting and immuno-fluorescence detection of Hsp70 were carried out. Results: Results showed elevated levels of Hsp70 mRNA and protein in SG cells of larvae immediately after gamma radiation exposure. The levels dropped to basal values by 48 h in the recovery period. Conclusions: The present study confirmed that radio-tolerant midge, C. ramosus expressed Hsp70 upon gamma radiation exposure and Hsp70 might be one of the gamma radiation-induced stress proteins required during the early stages of radiation stress management in aquatic midge larvae. This is the first report of its kind from the juvenile stage of any aquatic insect group.

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.

Characterization of highly toxic indigenous strains of mosquitocidal organism Bacillus sphaericus

Three indigenous isolates of Bacillus sphaericus (ISPC-5, ISPC-6 and ISPC-8), along with standard 2362 and 1593 strains, were evaluated for spore viability and mosquitocidal activity. Among these, ISPC-8 was the most viable and virulent isolate, exhibiting a significantly higher total viability count (TVC) and lower LC(50) values. The TVC of the standard strains ranged from 4.0 to 9.2 x 10(8) spores mL(-1), whereas it was 1.3 x 10(9) spores mL(-1) for ISPC-8. The LC(50) values of ISPC-8, 2362 and 1593 against Culex quinquefasciatus were 0.68 x 10(3), 1.22 x 10(3) and 1.85 x 10(3) spores mL(-1), respectively. The ISPC-8 was further assessed for host spectrum and found to be more active against C. quinquefasciatus, followed by Culex tritaeniorhynchus, Aedes albopictus and Aedes aegypti. The ISPC-8 strain was thus found to be a promising isolate for developing biopesticides. Among the indigenous strains, only ISPC-8 was found to have binary toxin genes (binA and binB). Comparative sequence analysis revealed that the BinA (41.9 kDa) protein of ISPC-8 differs by one amino acid (R197M), whereas BinB (51.4 kDa) differs by two amino acids (H99P, P174S) as compared with 1593 and 2362 strains. The purified binary proteins of ISPC-8 showed an LC(50) value of 6.32 ng mL(-1) against C. quinquefasciatus larvae after 48 h.

Gamma radiation tolerance of a tropical species of midge, Chironomus ramosus Chaudhuri (Diptera: Chironomidae).

Purpose: The Chironomid midges are known to thrive well under adverse environmental conditions and are even found inhabiting in areas contaminated by radioactive wastes. Studies were therefore undertaken to find out the radiosensitivity of different developmental stages of the Indian tropical midge, Chironomus ramosus. Materials and methods: In order to determine the threshold levels of lethality, eggs, larvae, pupae and adults of C. ramosus were exposed to varying dosages of gamma radiation (60Co radiation source) ranging from 0–3500 Gray (Gy) at dose-rate of 5.5 Gy/minute. The post-irradiation studies were conducted at three different time points: (a) Immediately after the end of irradiation, (b) 24 hours (h), and (c) 48 h after the end of radiation treatments. Determination of the lethal dose required to kill 50% (LD50), 90% (LD90) and 100% population was carried out using the log-probit analysis. Results: Different developmental stages showed variable threshold levels of radiosensitivity. The radiation doses required to cause 100% mortality immediately after radiation exposure of egg, larva, pupa and adult stages were 1000 Gy, 3000 Gy, 3200 Gy and 3500 Gy, respectively, indicating eggs as the most sensitive stage. Detailed analysis of the LD50 values of different post-irradiation time points indicated that pupal stages were also sensitive at 48 h post-irradiation amongst all the post-embryonic stages as described in many other insects. Interestingly detailed analysis of data indicated that amongst the adult population, females were the most radioresistant, compared to the males as reported in many other insect groups in the literature. Conclusions: The Indian tropical midge C. ramosus was found to tolerate higher dose of gamma radiation as compared to other known dipteran insects. It is evident from the present findings that C. ramosus falls in the category of radiation-tolerant group of insects.

Expression, purification and characterization of the Cry2Aa14 toxin from Bacillus thuringiensis subsp. kenyae.

An indigenous strain HD-550 of Bacillus thuringiensis subsp. kenyae was found to be toxic to lepidopteran as well as dipteran insects. The cry2Aa gene (classified as cry2Aa14) from this isolate was cloned and expressed in Escherichia coli. Only a little amount of the expressed Cry2Aa14 protein was observed in soluble fraction under normal induction condition. The inclusions were non-toxic to test insects, whereas solubilized Cry2Aa14 was highly toxic to lepidopteran and dipteran insects. Cry2Aa14 protein was expressed as thioredoxin (trx) fusion protein for improving the yield of active protein. An enhancement of nearly 15% was observed in the yield of active Cry2Aa14. The TrxA-Cry2Aa14 protein purified from the solubilized fraction also showed toxicity profile similar to the wild-type protein. The LC(50) values of Cry2Aa14 and TrxA-Cry2Aa14 protein against Spodoptera litura was 694 and 696 ng/cm(2), respectively, while for Culex quinquefasciatus the LC(50) values were 894 and 902 ng/ml, respectively. The broad spectrum toxicity of the Cry2Aa14 thus indicates that this protein could be an important component in integrated pest management. Further, the trx tag clearly led to higher yield, which facilitates protein purification for biophysical and biochemical characterization.